Dichotomophthora

Mehrl. & Fitzp. ex M.B. Ellis, Dematiaceous Hyphomycetes (Kew): 388. 1971. Fig. 12.

Fig. 12. Dichotomophthora lutea. A–E. Colonies on PDA. F–J. Colonies on OA. K. Habit. L–N. Conidiophores and conidia. O–Q. Conidiogenous cells. R, S. Conidiogenous cells and conidia. T, U. Conidia and microconidia. V–X. Sclerotia. Y. Anastomosing conidia. (A, F = ex-type CBS 145.57; B, G, K, O–T, V, Y = CBS 584.71; C, H = CBS 585.71; D, I, U = CBS 132.81; E, J, W, X = CBS 518.78). Scale bars: V–X = 100 μm; L, P = 20 μm; others = 10 μm.

Synonyms: Dichotomophthora Mehrl. & Fitzp., Mycologia 27: 550. 1935. (nom. inval., Art. 39.1, Melbourne).

Dichotomophthora Mehrl. & Fitzp. ex P.N. Rao, Mycopath. Mycol. appl. 28: 139. 1966. (nom. inval., Art. 39.1, Melbourne).

Classification: Dothideomycetes, Pleosporomycetidae, Pleosporales, Pleosporaceae.

Type species: Dichotomophthora portulacae Mehrl. & Fitzp. ex M.B. Ellis. Type specimen and ex-paratype strain: IMI 8742, CBS 174.35.

DNA barcodes (genus): ITS.

DNA barcodes (species): ITS, rpb2, gpdh. Table 5. Fig. 13.

Table 5. DNA barcodes of accepted Dichotomophthora spp.

Species Isolates1 GenBank accession numbers2 References
ITS gapdh rpb2
Dichotomophthora basellae CPC 33016T LT990654 LT990670 LT990640 Present study
Di. brunnea CBS 149.94T LT990653 LT990669 LT990639 Present study
Di. lutea CBS 145.57T LT990647 LT990663 LT990634 Present study
CBS 584.71 LT990648 LT990664 LT990635 Present study
CBS 585.71 LT990649 LT990665 LT990636 Present study
CBS 518.78 LT990650 LT990666 Present study
CBS 132.81 LT990651 LT990667 LT990637 Present study
Di. portulacae CBS 174.35PT LT990652 LT990668 LT990638 Present study
1

CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute. T and PT indicate ex-type and paratype strains, respectively.

2

ITS: internal transcribed spacers and intervening 5.8S nrDNA; gapdh: partial glyceraldehyde-3-phosphate dehydrogenase gene; rpb2: partial RNA polymerase II second largest subunit gene.

Fig. 13. RAxML phylogram obtained from the combined ITS (759 bp), LSU (880 bp), gapdh (594 bp) and rpb2 (958 bp) sequence alignment of all the accepted species of Dichotomophthora. The tree was rooted to Curvularia portulacae CBS 239.48 and BRIP 14541. The novel species described in this study are shown in bold. RAxML bootstrap support (BS) values above 70 % are shown in the nodes. GenBank accession numbers are indicated in Table 5. T and PT indicate ex-type and ex-paratype strains, respectively. TreeBASE: S21899.

Conidiophores macronematous, mononematous, unbranched or irregularly branched, sometimes swollen and repeatedly dichotomously or trichotomously branched or lobed at apex, forming a stipe and head; stipe hyaline to brown; branches usually short. Conidiogenous cells mono- or polytretic, integrated, terminal, lobed, cicatrized. Conidia solitary, dry, simple, ellipsoidal to cylindrical, rounded at ends, subhyaline to brown, multi-distoseptate. Microconidia ovoid, 0–2-distoseptate. Sclerotia often formed in culture resembling immature perithecia, semi- or immersed in agar, subglobose, ellipsoidal, ovoid, dark brown or black. Sexual morph unknown (adapted from Ellis 1971).

Culture characteristics: Colonies on PDA and OA white, hazel, orange, or dark grey to olivaceous, cottony, velvety, somewhat fluffy, or flat, margin irregular, effuse; reverse centre hazel, dark brown, periphery hazel, orange to luteous. Diffusible pigment luteous to orange (produced in some strains).

Optimal media and cultivation conditions: On PDA and OA at 25 °C under near-ultraviolet light (12 h light, 12 h dark). Some strains are sterile in culture.

Distribution: Worldwide.

Hosts: Anredera and Basella (Basellaceae), Beta vulgaris (Chenopodiaceae), Gymnocalycium mihanovichii var. friedrichii and Myrtillocactus geometrizans (Cactaceae), Portulaca (Portulacaceae), and soil.

Disease symptoms: Leaf spots, foliar abscission, stem blight, seed rot and damping-off.

Notes: Dichotomophthora was introduced as a monotypic genus with Di. portulacae isolated from Portulaca oleracea in Hawaii (Mehrlich & Fitzpatrick 1935). However, the publication lacked a Latin diagnosis of the fungus and the name was therefore invalid. Later, Rao (1966) provided a Latin description of Di. portulacae and introduced a new species, Di. indica, but the validation was misapplied and both names were regarded as invalid (de Hoog & van Oorschot 1983). Ellis (1971) validated the genus and the species based on the holotype specimen of Di. portulacae (IMI 8742). De Hoog & van Oorschot (1983) revised the taxonomy of Dichotomophthora and included Di. portulacae and Di. lutea. Dichotomophthora portulacae was restricted to species with dichotomously branched conidiophores, and conidia with 2–3 septa, 45–75 × 20–30 μm. Dichotomophthora lutea was introduced based on Dactylaria lutea, which is characterised by unbranched or irregularly branched conidiophores, and conidia with 1–5 septa, 30–115 × 10–20 μm.

 Dichotomophthora species are mainly known as plant pathogens with a wide host range as well as soil-borne fungi or saprobes (Mehrlich and Fitzpatrick, 1935, Routien, 1957, Rao, 1966, Ellis, 1971, Klisiewicz, 1985, Baudoin, 1986, Pfeiffer et al., 1989, Eken, 2003, Farr and Rossman, 2017, Soares and Nechet, 2017). However, a case of human keratitis caused by Di. portulacae was reported from subtropical Africa (de Hoog et al. 2000). Since many records of Di. portulacae may represent Di. lutea due to previous taxonomic confusion, host and distribution data need to be re-evaluated (de Hoog & van Oorschot 1983, Farr and Rossman, 2017, Soares and Nechet, 2017).

 This is the first time that numerous isolates, including the ex-type strains of both species of Dichotomophthora, have been subjected to phylogenetic analyses. Our results suggest that Dichotomophthora belongs in the Pleosporaceae (Pleosporales), closely related to Curvularia. The phylogenetic analysis and subtle morphological evidence revealed two additional new species, introduced here as Di. basellae and Di. brunnea. For an accurate identification at the species level, a DNA sequence analysis is recommended, since Dichotomophthora species are morphologically variable in culture and on natural substrates.

References: Mehrlich and Fitzpatrick, 1935, Routien, 1957, Rao, 1966, Ellis, 1971, de Hoog & van Oorschot 1983 (taxonomy and morphology); Klisiewicz, 1985, Baudoin, 1986, Pfeiffer et al., 1989, Eken, 2003, Soares and Nechet, 2017 (pathogenicity).


Dichotomophthora basellae

Hern.-Restr., Cheew. & Crous, sp. nov. MycoBank MB824604. Fig. 14.

Fig. 14. Dichotomophthora basella (ex-type CPC 33016). A–C. Disease symptoms caused by Di. basella in leaves of Basella rubra. D. Colony on PDA. E. Colony on OA. F. Colony overview with sclerotia and conidiophores. G–I. Conidiophores and conidia. J, K. Conidiogenous cells. L. Conidia. M. Microconidia. N, O. Sclerotia. Scale bars: N, O = 100 μm; H = 50 μm; others = 10 μm.

Etymology: Name reflects the substrate from which this fungus was isolated, Basella alba.

Hyphae 3–7.5 μm wide, hyaline to brown, septate, smooth to verruculose. Conidiophores macronematous, mononematous, unbranched or irregularly branched, sometimes swollen and repeatedly dichotomously or trichotomously branched or lobed at apex, forming a stipe and head; stipe 970–1370 × 10–12(–14) μm, pale brown, smooth; branches usually short; head 23–65 μm wide, pale brown to brown. Conidiogenous cells polytretic, integrated and terminal, lobed, cicatrized, individual lobes 6–14 × 6–9.5 μm. Conidia 32–86 × 10–18 μm, solitary, dry, ellipsoidal to cylindrical rounded at ends, subhyaline to yellow brown, 2–5-distoseptate. Microconidia 11–30 × 9–13(–15) μm, obovoid to ellipsoidal, 0–2-distoseptate. Sclerotia 295–444 × 234–409 μm, resembling immature perithecia, semi- or immersed in agar, globose, subglobose, ellipsoidal, ovoid, dark brown or black. Sexual morph unknown.

Culture characteristics: Colonies at 25 °C under near-ultraviolet light (12 h light, 12 h dark), on PDA and OA reaching 45–50 mm after 1 wk, centre black, periphery luteous, velvety, flat, margin regular, effuse; reverse centre olivaceous, dark brown, periphery pale luteous. Diffusible pigment luteous.

Material examined: Thailand, Chiang Mai, Chiang Mai university experimental farm, on leaves of Basella alba (Basellaceae), 2010, R. Cheewangkoon (holotype CBS H-23383, culture ex-type CPC 33016).

Notes: Dichotomophthora basellae is represented by one strain isolated from leaf spots on Basella rubra in Thailand. This species is morphologically similar to Di. lutea in having multi-lobed conidiogenous cells producing pale brown conidia, and colonies that produce a luteous to orange diffusible pigment in culture. In our study, Di. basellae produces larger conidia than Di. lutea (32–86 × 10–18 μm vs. 14–65.5 × 7.5–13 μm). However, de Hoog & van Oorschot (1983) described larger conidia in Di. lutea (30–115 × 10–20 μm). Additional studies with more isolates are thus required to compare these morphological differences and substrate preferences of both species.


Dichotomophthora brunnea

Hern.-Restr. & Crous, sp. nov. MycoBank MB824605. Fig. 15.

Fig. 15. Dichotomophthora brunnea (ex-type CBS 149.49). A. Colony on PDA. B. Colony on OA. C. Colony overview with conidiophores. D–J. Conidiophores, conidiogenous cells and conidia. K. Microconidia. L–Q. Conidia. Scale bars: D–G = 50 μm; H = 20 μm; others = 10 μm.

Etymology: From the Latin brunnea meaning brown, because of the brown colour of the conidia.

Hyphae 2.5–7 μm wide, hyaline to dark brown, septate, slightly constricted at septa, smooth. Conidiophores macronematous, mononematous, repeatedly dichotomously or irregularly branched, lobed at apex, forming a stipe and head; stipe 42–536 × 4.5–7.5 μm, pale brown to brown, smooth; branches usually short; head 10–28 μm wide, brown to pale brown. Conidiogenous cells mono- or polytretic, integrated, terminal, lobed, cicatrized, individual lobes 6.5–17 × 4–9 μm. Conidia 29–56.5 × 6–10 μm, solitary, dry, ellipsoidal to cylindrical rounded at ends, brown to dark brown, 2–6(–8)-distoseptate, straight or slightly curved. Microconidia 13–19.5 × 7–9.5 μm, obovoid to ellipsoidal, 0–1-distoseptate. Sclerotia not observed. Sexual morph unknown.

Culture characteristics: Colonies on PDA and OA at 25 °C under near-ultraviolet light (12 h light, 12 h dark), after 1 wk, reaching 90 mm, dark olivaceous, velvety, margin irregular, rhizoid (PDA) or entire (OA); reverse black. Diffusible pigment not produced.

Material examined: Unknown country, unknown substrate, date and collector (holotype CBS H-23382, culture ex-type dep. A. Arambarri LPS 325 = CBS 149.94).

Notes: Dichotomophthora brunnea was previously maintained as Di. portulacae in the CBS collection. However, the phylogenetic analysis suggests that the strain CBS 149.94 is a distinct species. Morphologically, the new species differs from Di. basellae and Di. lutea in having dark brown conidia and conidiogenous cells with 1–3 lobes (vs. yellowish or pale brown conidia and conidiogenous cells with usually more than three lobes). Neither pigment nor sclerotia were observed on the media tested.

Dichotomophthora lutea (Routien) de Hoog & Oorschot, Proc. Kon. Ned. Akad. Wetensch., Sect. C 86: 56. 1983. Fig. 12.

Basionym: Dactylaria lutea Routien, Mycologia 49: 191. 1957.

Synonym: Dichotomophthora indica Rao, Mycopath. Mycol. Appl. 28: 139. 1966. (nom. inval., Art. 35.1, Melbourne).

Hyphae 3–7.5 μm wide, hyaline to brown, septate, smooth to verruculose. Conidiophores macronematous, mononematous, unbranched or irregularly branched, sometimes swollen and repeatedly dichotomously or trichotomously branched or lobed at apex, forming a stipe and head; stipe 7.5–10(–12) μm wide, hyaline to brown, smooth; branches usually short; head 16.5–62 μm wide, pale brown to brown. Conidiogenous cells polytretic, integrated and terminal, discrete, lobed, cicatrized, individual lobes 8–13 × 4–11 μm. Conidia 14–65.5 × 7.5–13 μm, solitary, dry, ellipsoidal to cylindrical rounded at ends, straight to slightly curved, subhyaline to yellow brown, 0–4-distoseptate, sometimes constricted at septa, sometimes anastomosing conidia observed. Microconidia 12–27 × 7–13 μm, obovoid, 0–2-distoseptate. Sclerotia 146–325 × 197–370 μm, present or absent, often formed in culture, resembling immature perithecia, semi- or immersed in agar, globose, subglobose, ellipsoidal or ovoid, dark brown or black. Sexual morph unknown.

Culture characteristics: Colonies on PDA and OA at 25 °C under near-ultraviolet light (12 h light, 12 h dark), reaching 20–80 mm after 1 wk, white, hazel, orange, or dark grey to olivaceous, cottony, velvety, somewhat fluffy, or flat, margin irregular, effuse; reverse centre hazel, dark brown, periphery hazel, orange to luteous. Diffusible pigment luteous to orange (produced in some strains).

Materials examined: Argentina, isolated from soil, unknown date, J.B. Routien, (culture ex-type of Dactylaria lutea CBS 145.57). Cuba, Santiago de las Vegas, on leaves of Portulaca oleracea (Portulacaceae), 9 Mar. 1980, G. Arnold, INIFAT A80/85 = CBS 132.81. Italy, isolated from seedbed of Pinus radiata (Pinaceae), unknown date, G. Magnani, CBS 584.71. The Netherlands, on leaves of Portulaca oleracea (Portulacaceae), unknown date and collector, CBS 585.71; The Hague, on leaves of Portulaca oleracea (Portulacaceae), Jul. 1978, G.H. Boerema, CBS 518.78.

Notes: In the phylogenetic tree (Fig. 13), Di. lutea is represented by five strains isolated from soil and leaves of Po. oleraceae from Argentina, Cuba, Italy and the Netherlands. This species shows morphological variation among strains, with different colour and aspect of the colonies, production or absence of diffusible pigment and sclerotia. The above description is based on CBS 584.71, CBS 585.71, CBS 518.78 and CBS 132.81. The conidia were smaller than those described by de Hoog & van Oorschot (1983) based on the ex-type strain CBS 145.57 (14–65.5 × 7.5–13 vs. 30–115 × 10–20 μm). Unfortunately, the ex-type strain was sterile under the culture media and conditions tested.


Dichotomophthora portulacae

Mehrl. & Fitzp. ex M.B. Ellis, Dematiaceous Hyphomycetes (Kew): 388. 1971.

Synonyms: Dichotomophthora portulacae Mehrl. & Fitzp., Mycologia 27: 550. 1935. (nom. inval., Art. 39.1, Melbourne).

Dichotomophthora portulacae Mehrl. & Fitzp. ex P.N. Rao, Mycopath. Mycol. Appl. 28: 139. 1966. (nom. inval., Art. 38.5(a), Melbourne).

Hyphae 1.5–6 μm wide, subhyaline to pale brown, septate, slightly constricted at septa, smooth to verruculose. Conidiophores macronematous, mononematous, branched more or less dichotomously in apical region, forming a stipe and head; stipe 120–220 μm long, up to 14 μm wide at apex, reddish brown, smooth, verruculose near base, terminal branches up to 110 μm long, each ending in two slightly swollen, rounded or angular lobes. Conidiogenous cells mono- or polytretic, integrated and terminal, cicatrized. Conidia 45–75 × 20–30 μm, solitary, dry, ellipsoidal to cylindrical rounded at ends, dark reddish-brown, smooth, 2–3-distoseptate. Sclerotia 120–170 μm diam, often formed in culture, resembling perithecia, subglobose to globose or ovoid, dark reddish-brown. Sexual morph unknown (adapted from de Hoog & van Oorschot 1983).

Culture characteristics: Colonies at 25 °C under near-ultraviolet light (12 h light, 12 h dark) after 1 wk, on PDA reaching 60 mm, centre white, periphery olivaceous, cottony, margin irregular, effuse, white; reverse centre black, periphery olive. On OA reaching 45 mm, pale greenish grey, cottony, margin effuse, buff; reverse greenish olivaceous. Diffusible pigment not produced.

Material examined: USA, Hawaii, on Portulaca oleracea (Portulacaceae), unknown date and collector, isol. F.P. Mehrlich (ex-paratype culture CBS 174.35).

Notes: In this study Di. portulacae was represented only by the ex-paratype strain, which together with Di. brunnea (CBS 149.94) formed a subclade in Dichotomophthora. Both species have dark brown or reddish brown conidia and conidiogenous cells with 1–3 lobes. Nevertheless, Di. portulacae has shorter conidia with fewer septa, (45–75 × 20–30 μm, 2–3-distoseptate vs. 29–56.5 × 6–10 μm, 2–6-distoseptate).

Authors: M. Hernández-Restrepo, R. Cheewangkoon & P.W. Crous